Do Seed Vlcfas Trigger Spongy Tissue Formation in Alphonso Mango by Inducing Germination?

Do seed VLCFAs trigger spongy tissue formation in Alphonso mango by inducing germination?

SESHADRI SHIVASHANKAR*, MANOHARAN SUMATHI and TAPAS KUMAR ROY Division of Plant Physiology and Biochemistry, Indian Institute of Horticultural Research, Bangalore, India

*Corresponding author (Email, [email protected])

Spongy tissue is a physiological disorder in Alphonso mango caused by the inception of germination-associated events during fruit maturation on the tree, rendering the fruit inedible. Inter-fruit competition during active fruit growth is a major contributing factor for the disorder which leads to reduced fat content in spongy tissue affected fruits. This study was, therefore, carried out to determine the possible association between seed fats and ST formation. The study of the fat content during fruit growth showed that it increased gradually from 40% fruit maturity. At 70% maturity, however, there was a sudden increase of fat content of whole fruit, leading to acute competition and resulting in differential allocation of resources among developing fruits. As a result, the seed in spongy-tissue-affected mature ripe fruit showed a marked drop in the levels of fats and the two very long chain fatty acids (VLCFAs), tetracosanoic acid and hexacosanoic acid together with an increase of linolenic acid and a fall in oleic acid contents, which are known to be key determinants for the initiation of pre-germination events in seed. Subsequently, a rise in the level of cytokinin and gibberellins in ST seed associated with a fall in abscisic acid level clearly signalled the onset of germination. Concurrently, a significant reduction in the ratio of linolenic acid/linoleic acid in pulp led to the loss of membrane integrity, cell death and the eventual formation of spongy tissue. Based on the above, it is concluded that a significant reduction in the biosynthesis of VLCFAs in seeds during fruit growth might trigger pre-germination events followed by a cascade of biochemical changes in the pulp, leading to lipid peroxidation and membrane injury in pulp culminating in ST development. Thus, this study presents crucial experimental evidence to highlight the critical role played by VLCFAs in inducing ST formation in Alphonso mango during the pre-harvest phase of fruit growth.

[Shivashankar S, Sumathi M and Roy TK 2015 Do seed VLCFAs trigger spongy tissue formation in Alphonso mango by inducing germination? J. Biosci. 40 375–387] DOI 10.1007/s12038-015-9515-7

1. Introduction pre-harvest phase resulting in the development of ST in ripe fruits (Shivashankar 2014). Spongy tissue (ST) is a physiological disorder character- In order to explain the mechanism of ST formation in ized by the presence of off-white sponge-like pulp tis- only some and not all fruits on a panicle, it was as- sue, slightly desiccated, with or without air pockets, sumed that due to temporal variations in fertilization of originating on the surface of the stony pit, resulting in flowers and fruit set, developing mango fruits at any the production of poor quality fruit (Katrodia 1979). given time are likely to be at different physiological This disorder adversely affects the sensory quality of stages of maturity and are subject to variable competi- fruits without showing external symptoms. The primary tion during their ontogeny (Shivashankar 2014). Further, cause of this disorder was first established in our studies it was postulated that the genetically stronger sinks which showed that the Alphonso mango seed switches which sustain the competition grow to full maturity over to germination phase during fruit maturation in the producing healthy fruits while the seed from weaker

Keywords. Alphonso mango; cytokinin synthesis; membrane damage; seed germination; spongy tissue; VLCFA http://www.ias.ac.in/jbiosci J. Biosci. 40(2), June 2015, 375–387, * Indian Academy of Sciences 375

Published online: 17 April 2015 376 S Shivashankar, M Sumathi and TK Roy sinks which cannot sustain the competition get separated 2.2 Fat content from the mother plant physiologically as the vascular strands (funiculus) between the peduncle and endocarp Total fat content in tissue samples was extracted using (stone) become disconnected, even while on the tree Soxhlet extractor as described by Osborne and Voogt (Wainwright and Burbage 1989), causing a physiological (1978) and estimated gravimetrically. One gram of dry tissue shock to the embryo, thus affecting its dormancy or powder packed in a thimble was placed in a Soxhlet extrac- viability leading to seed germination in the developing tor and extracted under reflux on a steam bath for 3 hrs using fruit. Previous studies in our laboratory had established petroleum ether as solvent (bp 40°–60°C). The solvent con- that the process of seed-germination-associated events taining the dissolved fats was quantitatively transferred to a led to water uptake from the surrounding mesocarp, dry pre-weighed flask (W1), evaporated to dryness on a resulting in the breakdown of seed reserves followed boiling water bath and weighed again (W2). The difference by changes in pulp composition due to which changes in the weight was used to calculate the percent of fat in occurred in texture and sensory properties compared to sample using the formula, (W2−W1)/Weight of sample×100. healthy normal tissue (Shivashankar et al. 2007; Ravindra and Shivashankar 2004) and resulted in spongy 2.3 GC-FID analysis tissue formation. However, the all-important question as to what triggered the seed germination process in some Healthy and spongy affected fruit tissues were homogenized fruits leading to damage to cell membrane and the in a mixture of chloroform-methanol (2:1 v/v) and filtered biochemical mechanism of development of ST had not through Whatman no.1 filter paper. The chloroform phase been established. containing the lipids was separated, dried in a rotary vacuum Incidentally, Alphonso mango fruit is reported to contain evaporator at 40°C and stored at −20°C until further used the highest percentage of fats in both pulp and seed com- (Folch et al. 1957).The extracted lipids were methylated by pared to other mango varieties (Selvaraj 1996). Past work dissolving in methanol and refluxing for 10 min at 70°C, had shown that the ST-affected Alphonso fruit contained less followed by addition of 14% BF3 in methanol and further fats compared to healthy fruit (Shivashankar et al. 2007). refluxed for 30 min at 70°C according to the modified This suggested a possible link between fat content and method of Morrison and Smith (1964). Methyl esters of fatty sponginess formation. Since the biosynthesis of fats is an acids (FAME) were subsequently extracted in heptane and energy-intensive process, we speculated that under condi- dried on anhydrous sodium sulfate and filtered through tions of limiting assimilate supply during fruit development 0.2 μm nylon membrane. on the tree, synthesis of fats might be inhibited in some GC-FID analysis of fatty acid methyl esters was carried fruits, resulting in such fruits turning spongy after ripening. out using a Varian-3800 Gas chromatograph system A review of literature suggested that, in spite of extensive equipped with flame ionization detector ( FID) on a fused studies on the subject of spongy tissue by several investiga- silica capillary column (VF-5 Factor Four, Lake Forest, CA, tors around the world thus far, there was no attempt to USA), 30 m × 0.25 mm i.d. and 0.25 μm film thickness. The connect fatty acid metabolism in seed and pulp to the process temperature program for the column was as follows: The of ST formation in Alphonso mango. This study was, there- initial oven temperature was 100°C for 4 min, increased by fore, carried out with the aim of examining the possible role 3°C per min up to 220°C, held for 4 min, temperature of seed fats and fatty acids in ST formation. Based on the increased further at the rate of 5°C per min up to 260°C results of the study, the sequence of biochemical events and held for 10 min. Injector and detector temperatures were leading to cell membrane injury and the eventual formation maintained at 250°C and 260°C respectively. Helium was of spongy tissue in Alphonso mango fruit are discussed in used as the carrier gas at a flow rate of 1 mL/min. Flow rates this paper. of H2 and air were maintained at 20 mL/min and 250 mL/ min respectively. Initially injection mode was split-less followed by split mode (1:30) after 1.5 min. 2. Materials and methods 2.3.1 Gas chromatography–mass spectrometry (GC-MS): GC- 2.1 Tree growth conditions MS analysis was performed on Varian-3800 gas chromatograph coupled with Varian 4000 GC-MS-MS ion trap mass selective ‘Alphonso’ mango fruits were collected during the 2012– detector. Fatty acids were separated on VF-5MS fused silica 2013 season from 25-year-old trees receiving the recom- capillary column (Varian, USA) (30 m × 0.25 mm i.d. with mended supply of fertilizers and plant protection measures 0.25 μm film thickness) by applying the same temperature pro- and maintained under uniform growth conditions in the gram as described above for GC-FID analysis. The carrier gas experimental orchard of IIHR, Bangalore. was helium at a flow rate of 1mL/min; injector temperature,

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260°C; ion source-temperature, 220°C; trap temperature, 200°C all the above components was also maintained. The reaction and transfer line temperature, 260°C. Mass detector conditions was terminated by adding 0.5 mL dinitro salicylic acid were: EI-mode at 70 eV with full scan range, 50–450 amu. (DNS) reagent and heated for 5 min on a boiling water bath. Fatty acids were identified by comparing the relative The mixture was made up to a final volume of 20 mL and its retention times of FAME peaks with those of reference absorbance was read at 540 nm (Bernfeld 1955). α-Amylase standards (Sigma-Aldrich, USA) and also by comparing activity was expressed as mg maltose liberated/g/h the spectra with those available in Wiley and NIST-2007 spectral libraries (Liu 1994). The total quantity of FAME 2.5.3 Malate dehydrogenase: Malate dehydrogenase (MDH) was estimated as the sum of all GC-FID peak areas in the activity was determined as described by Selvaraj et al.(1995). chromatogram and individual compounds were quantified by The assay mixture consisted of 0.4 mL 0.2 M Tris-HCl buffer, comparing the known individual FAME procured as stan- pH 7.5, 0.5 mL 1 mM NADH, 0.5 mL 15 mM oxaloacetate, dard. All the analyses were performed on three samples run and 0.1 mL enzyme extract. Absorbance at 340 nm was separately. measured at 30°C, and MDH activity was expressed as change in absorbance at 340 nm/mg protein/min. 2.4 Intensity of spongy tissue 2.5.4 Succinate dehydrogenase: Succinate dehydrogenase Intensity of spongy tissue was quantified as a percentage of (SDH) was assayed in a reaction mixture containing 0.45 mL the fresh weight (FW) of spongy-tissue-affected pulp com- 0.5% (w/v) 2,3,5 triphenyltetrazolium chloride (TTC), 0.55 pared to the total FW of the pulp. The spongy tissue affected mL 0.2 M sodium succinate, 1.3 mL 0.1 M sodium phosphate spot showing discoloured and partially dehydrated tissue buffer, pH 7.2 and 0.2 mL enzyme extract, in a total volume of “ ” was considered ST-affected (ST) and the tissue surround- 2.5 mL. The enzyme activity was expressed as the change in ing the affected spot which remained free from symptoms absorbance at 460 nm/mg protein/min (Baqui et al. 1974). was designated as “apparently healthy” (AH). The healthy (H) tissue was collected from non-affected fruit. 2.5.5 Estimation of gibberellins, abscisic acid and cytokinins: Extraction and purification of seed hormones: Seeds (10 g) 2.5 Enzyme and phytohormone assays were ground in liquid nitrogen, mixed with 20 mL of 80:20 (v/v) mix of methanol and water containing 20 ng of each 2.5.1 Lipase activity: Lipase activity was determined as internal standard [Gibberellic acid (GA), abscisic acid (ABA) described by Jayaraman (1981). Five hundred milligrams and kinetin], and incubated at 4°C on a shaker at 300 rpm for of acetone powder was extracted with 10 mL of 0.05M 24 h in the dark. Samples were centrifuged at 300g for 10 min sodium phosphate buffer, pH 7.0 and left standing overnight at and the supernatant was transferred to a clean test tube. The o 4 C.The extract was centrifuged at 8000 rpm for 10 min and pellet was re-suspended in 5 mL of extraction solvent mix of supernatant was used as the enzyme source. The assay medium methanol: water [80:20 (v/v)] and centrifuged again at 300g consisted of 5 mL substrate (a mixture of 1 mL groundnut oil, for 10 min at 4°C. After centrifugation, the two supernatants 1.5 mL of 0.1 M phosphate-citrate buffer, pH 7.0, 0.5 mL of were combined and concentrated in a rotary flash evaporator at 10% gum Arabica and 5 mL water), 3 mL of 0.01M NaCl and 35°C, dissolved in double distilled water, pH adjusted to 3.0 2 mL of enzyme. The reaction mixture was incubated for and re-extracted three times with diethyl ether. The ether layer o 30 min at 37 C. The reaction was terminated after 30 min by was collected and mixed with sodium sulphate to remove the addition of 10 mL absolute alcohol. For the blank, the traces of moisture. The purified extract was concentrated under reaction was stopped at zero time. The reaction mixture was a continuous stream of nitrogen and the residue was re- titrated against N/200 NaOH using phenolphthalein indicator. dissolved in 200 μL HPLC-grade methanol, centrifuged at Lipase activity was expressed as mg free fatty acid (FFA) 12,740g for 10 min at 4°C, filtered through a 0.22-μmPTFE liberated /g/h based upon the standard graph constructed with filter (Waters, Milford, MA, USA) and separated by HPLC for linoleic acid as standard. the quantification of gibberellins and ABA. The aqueous layer obtained after separating the ether 2.5.2 Amylase activity: α-Amylase was extracted by homo- layer was adjusted to pH 8.0 and extracted three times with genizing 1.0 g of pulp in 10 mL of 16 mM sodium acetate water-saturated butanol. The butanol layer was collected and buffer, pH 4.8, containing 0.5 M NaCl and centrifuged at concentrated under a continuous stream of nitrogen. The 10,000g for 10 min at 4°C. Five-hundred μL of the super- residue was re-dissolved in 200 μL HPLC-grade methanol, natant was added to a reaction mixture containing 0.5 mL of centrifuged at 12,740g for 10 min at 4°C, filtered through a 1% (w/v) starch dissolved in the same extraction buffer and 0.22 μm PTFE filter (Waters, Milford, MA, USA) and used incubated for 30 min at 20°C. A zero time blank containing for separation of cytokinins.

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High-performance liquid chromatography (HPLC): The expressed as dS/ m. pH was recorded using a combination method of Kelen et al.(2004) with some modifications was electrode. used for identification and quantification of seed hormones by HPLC.Separation was performed using a Shimadzu LC- 2.9 Statistical analysis 10 AD VP liquid chromatograph, equipped with a stainless steel analytical column, (Synergi 4μ Hydro RP 80A; 250 mm ×4.6 mm id; Phenomenex, Torrance, CA, USA). Experimental data were subjected to ANOVA adapting the ’ Samples (10 μL) were injected into the HPLC system and Fisher s analysis of variance technique (Panse and Sukhatme separation of ABA and GA was carried out by isocratic 1978) and mean values were tested for significance using ’ elution using a 26:74 (v/v) mix of acetonitrile:water, pH Student s t-test. The results were expressed as mean ± stan- 4.0, as the mobile phase, at a flow rate of 0.8 mL/min. dard error (SE). Cytokinins were separated using a 14:86 (v/v) mix of acetonitrile:water containing 0.5%(v/v)acetic acid at a flow 3. Results rate of 0.2 mL/min. The mobile phase was filtered through a 0.22-μm nylon membrane and degassed before use. PGR 3.1 Rate of fat synthesis during fruit growth concentrations were monitored by recording the absorption at 200 nm, 220 nm and 270 nm for GA, ABA and cytokinins As shown in table 1, the levels of fats in both pulp and seed respectively and calculated using calibration curves prepared increased gradually with fruit maturity. However, at 70%, using 10 μL each of the standard (Sigma, St. Louis, MO, there was an abrupt increase in the rate of fat accumulation USA). by the fruit which continued until full maturity. A striking feature of the study was that there was a significant increase ≤ 2.6 Free radical production in the fat content of whole fruit (P 0.005) at 70% maturity at which time it was 7-times higher compared to 40% mature •– fruit and subsequently increased by about 15-times at 80% Superoxide anion (O ) levels were estimated following 2 maturity and 21-times at 90% maturity. Doke (1983). The levels of hydroxyl radicals (•OH) were determined as described by Von Tiedemann (1997), using 2- deoxyribose as the scavenger molecule. Hydrogen peroxide 3.2 Commencement of seed germination events content was measured according to Schopfer et al.(2001) and expressed as ng H2O2 generated /g FW of tissue. Lipase activity in seed of ST fruit was higher than the H fruit (P≤0.005) while on the contrary, pulp of ST fruit had lower lipase activity compared to H pulp. Lipase activity increased 2.7 Lipid peroxidation rapidly in seed with increasing intensity of ST (P≤0.005) while registering a rapid decline in pulp (figure 1). The Lipid peroxidation was monitored by measuring the conver- activities of amylase, SDH and MDH measured in seed were sion of lipids to malondialdehyde (MDA).using the thiobar- also higher in ST compared to H fruit (P≤0.005) while the bituric acid reactive substances (TBARS) assay, as described converse was true for pulp (figure 2). by Draper and Hadley (1990). TBARS reagent (1 mL) was The fat content of seed in mature healthy fruit was 8.5% added to a 0.5 mL aliquot of tissue homogenate and heated as against 7.7% in ST-affected fruit while fat content in pulp for 20 min at 100°C. The antioxidant, butylated hydroxy- of healthy fruit was 1.5% compared to 0.8% in ST-affected toluene, was added before heating the samples. After cooling fruit. The content of total free fatty acids was significantly on ice, samples were centrifuged at 840g for 15 min and lower in ST pulp and seed compared to healthy pulp and absorbance of the supernatant was read at 532 nm. Blanks seed (P≤0.005) respectively (table 2). for each sample were prepared and assessed in the same way to correct for the contribution of A532 to the sample. TBARS results were expressed as MDA equivalents using 1,1,3,3- 3.3 Generation of free radicals tetraethoxypropane as standard. As a first step towards analyzing the effect of seed germination on the possible changes in pulp, free radical production 2.8 Electrolyte leakage was estimated in healthy, apparently healthy (AH) and spongy tissue affected (ST) pulp. H2O2 levels showed a One gram of pulp tissue was suspended in 10 mL of distilled marked increase in AH (70.3%) and ST (114.8%) compared water and electrolyte leakage was measured as conductance to the control (P≤0.005). The level of hydroxyl radicals using a conductivity bridge (ELICO model CM-180) and (•OH) increased significantly in AH (587.6%) and further

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Table 1. Fat content1 of Alphonso mango during fruit growth

Parameter Fruit maturity (%) F- value

40 50 60 70 80 90 100

Pulp fat (g/100 g DW) 0.41±0.032 0.53±0.02 0.66±0.01 0.77±0.02 0.94±0.02 1.40±0.03 1.50±0.19 35.62 Seed fat (g/100 g DW) 1.60±0.15 2.48±0.19 4.00±0.21 5.95±0.22 7.40±0.27 8.00±0.34 8.50±0.76 65.53 Fruit fresh wt (g) 76.9±2.4 125.5±7.2 164.8±3.9 185.1±5.3 238.3±9.1 252.1±7.5 260.3±2.4 137.00 Total fat (g/FW fruit) 0.06±0.004 0.14±0.013 0.29±0.009 0.42±0.018 0.91±0.069 1.27±0.086 1.34±0.152 56.31

1Fat content in pulp and seed were extracted using Soxhlet extractor and estimated gravimetrically. Note the 7-, 15- and 21-fold increase of fat accumulation at 70, 80 and 90% fruit maturity. Respectively indicating increased competition for resources. 2 ± denotes standard error of mean. up in ST (1211.6%) compared to healthy control. Superox- acid, oleic acid and linoleic acid were lower in both pulp and ide radical (O2•–) levels also increased rapidly in AH seed of ST-affected fruit. There was a significant fall in oleic acid (167.6%) and shot up further in ST (404.4%) (figure 3). and the levels of the two very long chain fatty acids (VLCFA), tetracosanoic acid and hexacosanoic acid in both seed and pulp of ST-affected fruit while linolenic acid level increased in ST 3.4 Changes in free fatty acids and membrane seed with a steep fall in ST pulp. The levels of the two long chain degradation in pulp fatty acids, erucic acid and eicosanoic acid increased in ST pulp (table 3). The ratio of linolenic acid/linoleic acid decreased The influence of seed germination on the composition of significantly from 0.622 in healthy pulp to 0.059 in ST pulp fatty acids in pulp and membrane structure was examined. (P≤0.005) while the concentration of MDA in ST pulp (2.48 μg/ The levels of short and medium chain fatty acids, nonanoic 100 g FW) was higher compared to AH (1.64) and H tissue acid and lauric acid were higher in both seed and pulp of ST- (1.39). The pH of ST pulp was significantly lower (4.3) com- affected fruit compared to healthy fruit while the levels of pared to AH (4.9) and H pulp (5.4) (P≤0.05) while the EC was long chain fatty acids, palmitic acid, palmitoleic acid, stearic significantly higher in ST pulp (0.76) compared to AH (0.61) and H pulp (0.54) (P≤0.005) (figure 4).

3.5 Changes in the levels of cytokinin, gibberellins and abscisic acid in seed

The levels of cytokinins, abscisic acid (ABA) and gibberellins (GAs) in seed of healthy and ST-affected fruit are presented in figure 5. The concentrations of cytokinins namely, Zeatin(Z), Zeatinriboside (ZR), Dihydrozeatinriboside (DHZR), isopentenyl adenine(iP) and isopentenyl adenosine (iPA) were significantly higher in seed of ST fruit compared to H fruit. Gibber- ellins also increased significantly in seed of ST fruit while ABA content was lower in seed of ST fruit compared to H fruit.

4. Discussion Figure 1. Changes in lipase activity of Alphonso mango pulp as a function of intensity of ST (**P≤0.005). Fruits affected by increas- 4.1 Competition for resources leads to reduced fat ing intensity of ST were monitored for lipase activity by titrimetric estimation of the liberated free fatty acids. The rapid increase of synthesis activity in ST seed with increasing intensity of spongy tissue indicated the increased rate of conversion of stored fats during ST Therewasasteadyincreaseinthefatcontentofboth development. pulp and seed in developing fruits up to 60% fruit

J. Biosci. 40(2), June 2015 380 S Shivashankar, M Sumathi and TK Roy

From this account, it was evident that at 70% fruit maturity, the demand for supply of assimilates for fat synthesis in Alphonso fruit increased rapidly and continued to rise further until full maturation. Similar results were reported by Abou-Aziz et al. (1973)in avocado pear fruits supporting the present findings. Thus, it was apparent that 70% fruit maturity was the critical stage in Alphonso mango at which fruits on a panicle began to face strong competition for resources from neighbouring fruits for the synthesis of a greatly increased proportion of fats. Incidentally, the Alphonso mango fruit is reported to contain the highest fat content among all mango varieties examined so far (Selvaraj 1996) and is, therefore, thought to be a major sink for assimilates. Since carbohydrates provide the carbon skeleton for the synthesis of fats in plants (Kikuta 1969;Rawsthorne2002), it could be expected that a short supply of carbohydrates in Alphonso mango would lead to intense competition for resources among developing sinks at 70% maturity. Since dry matter partitioning among sinks is determined by the sink strength of various organs (Marcelis 1994, 1996), the allocation of assimilates to weak sinks would be reduced (Bertin 1995) due to acute competition among sinks as fruit growth in mango mainly occurs from the limited stored carbohydrate reserves of the previous year (Bustan et al. 1996). This presumably could result in reduced synthesis of fats in some fruits on a panicle. Data presented in table 2 confirmed this speculation, when it was found that the contents of total fats and free fatty acids in both seed and pulp of ST fruit were significantly lower compared to seed and pulp of healthy fruit respectively. The causal nature of reduced seed fat content leading to ST formation was clearly apparent when it was noted that some fruits of 80% maturity showing lower fat content (0.64–0.85%) than −1 −1 Figure 2. Activities of amylase (mg maltose liberated g h ), normal (0.91–1.27%) had higher seed moisture content −1 −1 −1 MDH ( A340 nm mg protein min ) and SDH (( A460 nm mg – −1 (42 54%) than normal (32%), a characteristic protein min ) enzymes in healthy and ST-affected pulp (A) and distinguishing feature of spongy-tissue-affected fruits. seed (B) of Alphonso mango fruit (**P≤0.005). The higher activities of amylase, MDH and SDH in ST seed associated Interestingly, the earliest stage at which the increase with a reduction in the pulp of ST confirmed the progress of of seed moisture was observed was at the 80% stage germinatin events in seed coupled with a reduced rate of starch of fruit maturity. This finding was of great significance conversion and generation of reducing power in pulp tissue as it provided positive proof for the fact that the flow during ST formation. of moisture from pulp to seed and the associated biochemical changes in the ST-affected seed were initiated around 70% fruit maturation stage although symptoms maturity. However, at 70% fruit maturity, there was a of the disorder became apparent only after fruit ripening rapid 7-fold increase in the fat content of whole fruit, (Shivashankar et al. 2007). Considering the fact that the which further increased to about 15-fold at 80% matu- onset of seed germination caused the ST disorder rity and further up to 21-fold at 90% maturity as (Ravindra and Shivashankar 2006), it was obvious that compared to 40% maturity fruits. The steep increase the reduction in the content of seed fat signalled the of fruit weight during the same period also contributed beginning of the sequence of events leading to ST for the marked increase of fat content of whole fruit. development in Alphonso mango.

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Table 2. Total fat, free fatty acids and lipase activity in pulp and seed of healthy (H) and spongy-tissue-affected (ST) Alphonso mango fruit

Parameter measured Pulp Seed

HSTt-Value H ST t-Value

Total fat1 (g/100 g FW) 1.5±0.33 0.8±0.20 5.14 8.5±0.27 7.7±0.20 6.69 Lipase activity3 (mg) fatty acid liberated/g/h) 71.5±2.1 48.6±3.9 13.32 65.6±3.5 76.0±2.7 −7.69 Free fatty acids2 (mg/100 g) 193.6±5.3 157.9±2.8 16.97 326.3±4.3 232.4±3.8 46.02

1 Note the marked reduction in the total fat content of both pulp and seed of ST fruit compared to H fruit showing a reduction in the synthesis of fat in ST fruit. 2 Free fatty acid composition determined by GC MS showed a significant reduction in both pulp and seed of ST fruit indicating that the synthesis of fatty acid was reduced in ST fruit. 3 Lipase activity in seed increased in seed indicating the initiation of seed germination while it reduced in pulp denoting slower degradation of fat in ST fruit pulp.

4.2 Changes in the fatty acid composition of seed fruit (figure 5).It is a well known fact that gibberellins and its effect on seed germination (GA) and cytokinins promote germination (Taylorson and Hendricks 1977; Nikolić et al. 2006) while, ABA is a From the data presented in table 3, it is worthwhile mention- potent inhibitor. Considering the changes in hormones ing that there was a significant decline in the levels of the and the activities of lipase (table 2), amylase and the two very long chain fatty acids (VLCFAs), tetracosanoic respiratory enzymes (figure 2) in ST seed, it was evident acid and hexacosanoic acid in seed from ST-affected fruit. that germination events had begun in ST seed. It was VLCFAs are fatty acids containing 20 to 36 carbons which thus apparent that a fall in the VLCFA content of seed perform a wide range of physiological functions depending could have provided the primary trigger for the onset of on their chain length and the level of unsaturation, making pre-germination events in seed as reported by Munshi them crucial for many vital processes such as cell expansion, et al. (2007). cell proliferation or differentiation (Bach and Faure 2010). Bach et al.(2008) showed that VLCFAs are essential for cell viability. High levels of VLCFAs in seed are known to 4.3 Effect of seed germination on pulp characteristics suppress biosynthesis of cytokinins (Nobusawa et al. 2013a) which are crucial for pre- and early post- Data presented in table 2 showed that the fat content, germination events (Letham and Bollard 1961; Miller FFA and lipase activity in pulp of ST-affected fruit were 1961; Fosket et al. 1977). On the contrary, a lower VLCFA lower compared to H fruit confirming that both the rate content in seed led to an increase of cytokinin level of synthesis and breakdown of fats were reduced in ST (Nobusawa et al. 2013b) which in turn countered the action pulp. Lipase activity in pulp decreased rapidly with the of germination inhibitors (Khan 1971, 1975) and overcame increasing intensity of ST in pulp (figure 1). The levels ABA-suppressed seed germination (Wang et al. 2011). of short and medium chain fatty acids, nonanoic acid, Based on these reports, it was apparent that a reduced level lauric acid and myristic acid increased in ST pulp, while of VLCFAs in ST seed would favour early germination. levels of long chain fatty acids, palmitic acid, palmitoleic Further, data presented in table 3 also showed a decrease in acid, stearic acid and oleic acid and the two VLCFAs, palmitic acid content coupled with an increase of linolenic tetracosanoic acid and hexacosanoic acid, registered a acid in ST seed compared to healthy seed. The analogous decline. Among the changes occurring in the FFA levels, changes in these two fatty acids in the embryonic axes of the increase in the contents of two long chain fatty acids, seeds have been correlated with faster germination in sun- erucic acid and eicosanoic acid in ST pulp were of flower (Munshi et al. 2007). Accordingly, changes in the significance as these fatty acids are known to exhibit levels of linolenic acid, palmitic acid and VLCFAs in ST higher transition temperatures, leading to decreased mem- seeds in comparison with the healthy seed clearly showed brane fluidity, permeability and stability of membrane the onset of pre-germination events in ST seed. (Bangham 1975). Additionally, another notable finding Further, measurement of the changes in the levels of was that while linoleic acid registered a marginal decline seed hormones showed a significant increase in the levels in ST pulp, linolenic acid level dropped markedly. Con- of cytokinins and gibberellins coupled with a reduction in sequently, the ratio of linolenic/linoleic acid in pulp re- ABA content of seed of ST-affected fruit compared to H duced significantly, indicating a higher rate of lipid

J. Biosci. 40(2), June 2015 382 S Shivashankar, M Sumathi and TK Roy

Figure 3. Pattern of free radical production in healthy, apparentlyb healthy and ST-affected Alphonso mango pulp (**P≤0.005). ST- affected pulp, pulp surrounding the ST-affected portion remaining free of ST symptoms and pulp from healthy fruit were analysed. Note the significant increase in free radical production, particularly the hydroxyl radical in AH and ST pulp compared to H pulp coupled with a drastic reduction in antioxidant enzyme activities, indicating rapid destruction of mesocarp tissue during ST formation. peroxidation. Earlier studies on Alphonso mango following seed germination concurrent with the climacteric ripening of fruit had indicated development of hypoxia in the pulp (Ravindra and Shivashankar 2004), which is also known to favour increased rate of lipid peroxidation (Blokhina et al. 2003).

4.4 Generation of free radicals and lipid peroxidation of membranes

The significantly higher levels of •OH and O2•– radicals generated in pulp of AH and ST compared to healthy tissue (figure 3) are considered to be highly detrimental as excessive levels of these two free radicals have been implicated in the destruction of plant cells through peroxidation (DaCosta and Huang 2007). Hydroxyl radical is one of the most destructive free radicals responsible for modifications of macromolecules and cellular damage (Jiang Ming-Yi 1999). As the level of free radicals increase, the rate of oxidation also increases unless the activity of antioxidant enzymes increases proportionately. In a previous report by Nagamani et al. (2010), the activities of anti-oxidative enzymes such as, catalase (CAT), peroxidase (POX) and superoxide dismutase (SOD), which together constitute a mutually supportive defence system against ROS, were found to be lower in ST compared to healthy tissue of Alphonso mango. A reduction in the activities of anti-oxidative enzymes was reported under hypoxic conditions (Ushimaru et al. 1997)as observed in ST pulp by the increased accumulation of CO2 (Ravindra and Shivashankar 2004). Considering the fact that SOD is involved in lowering the steady-state level of superoxide radicals while CAT and POX lower the level of H2O2 in higher plants, the greatly increased production of free radicals in ST pulp coupled with a reduction in the activities of anti- oxidative enzymes indicated increased peroxidation of lipids. Data presented in figure 4B showed that the level of MDA in ST pulp increased markedly compared to AH and H tissue.MDA is the end product of peroxidative decomposition of polyenic fatty acids in the lipid peroxidation process and its accumulation in tissues is indicative of the extent of lipid peroxidation. It was thus evident from the data that the production of a disproportionately higher level of free radicals coupled with lower activities of anti-oxidative enzymes

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Table 3. Fatty acid composition of healthy (H) and spongy-tissue-affected (ST) pulp of Alphonso mango fruit

Fatty acid Seed (mg/100 g) Pulp (mg/100 g)

Healthy Spongy t-Value Healthy Spongy t-Value

Nonanoic acid 2.37±0.12 2.77±0.23 −1.58 0.25±0.01 2.01±0.16 −25.57 Lauric acid 4.11±0.15 4.97±0.17 −3.62 0.99±0.09 6.28±0.37 −27.32 Myristic acid 0.48±0.07 0.35±0.07 1.50 0.21±0.01 0.29±0.10 −2.23 Palmitic acid 143.0±1.15 106.4±1.89 16.45 76.6±1.59 58.7±1.98 24.28 Palmitoleic acid 82.7±1.87 47.6±1.42 14.81 36.4±1.49 34.6±2.61 1.83 Stearic acid 46.3±1.97 33.4±0.88 5.93 32.7±1.15 26.9±2.78 4.74 Oleic acid 25.5±1.72 19.1±1.20 3.01 17.4±0.50 11.3±1.57 11.35 Linoleic acid1 8.73±1.14 7.48±1.02 0.85 3.99±0.36 2.67±0.31 6.81 Linolenic acid2 0.10 ±0.03 0.57±0.11 −3.98 2.48±0.18 0.16±0.03 30.13 Eicosanoic acid 1.79 ±0.16 1.84±0.17 −0.17 3.39±0.35 3.94±0.12 −4.17 Erucic acid 2.20±0.23 1.64±0.16 2.01 1.91±0.27 3.56±0.42 −9.67 Behenic acid 2.02±0.18 1.82±0.18 0.99 1.89±0.28 1.89±0.22 0 Tetracosanoic acid3 6.02±0.29 3.62±0.33 5.50 8.17±0.29 4.92±0.36 19.52 Hexacosanoic acid4 1.04±0.20 0.88±0.15 0.69 7.14±0.29 0.69±0.20 52.87

1,2 Note the marginal reduction in linoleic acid content and a significant reduction in the linolenic acid content of the ST seed which accounted for a major change in the ratio of linolenic acid to linoleic acid of the ST seed compared to H seed representing membrane injury in ST seed. 3,4 Note the marked reduction in the levels of the two VLCFA in ST seed triggering cytokinin biosynthesis and seed germination.

(Nagamani et al. 2010) contributed to a large accumulation of irreversible alteration to cell membranes (Dhindsa et al. malondialdehyde leading to increased rate of lipid peroxida- 1981). In the present study, this was evident from the in- tion in AH and ST pulp. Increase in lipid peroxidation has been creased EC of ST pulp (0.76) compared to AH (0.61) and H reported during leaf senescence, ozone injury, anoxia, drought pulp (0.54) while the pH was lower (4.3) compared to AH stress and wounding (Kepler and Novacky 1986). (4.9) and H pulp (5.4) (figure 4). Accordingly, it was apparent that increased lipid peroxidation in ST pulp led to loss of cell membrane integrity resulting in leakage of cellular con- 4.5 Changes in the linolenic/linoleic acid ratio tents. An acidic environment was thus created reducing the and membrane damage pH of the pulp, which, in turn, progressively damaged the surrounding tissues due to osmolysis, leading to death of As a consequence of increased lipid peroxidation, the ratio of cells. Scandalios (1993) reported that leakage of cellular linolenic/linoleic acid dropped significantly in ST pulp contents following peroxidation of plasmalemma leads to (figure 4A). The ratio of the two fatty acids, linolenic acid rapid desiccation and cell death. Light microscopic and linoleic acid is considered to be critical for membrane observations by Raymond et al.(1998) showing extensive structure and function. α-linolenic acid is reported to be 10 disintegration of walls of most cells in ‘Tommy Atkins’ and times more susceptible to oxidation than linoleic acid and, ‘Van Dyke’ mango fruits affected by jelly seed, a disorder therefore, readily undergoes conversion to the unstable hy- similar to spongy tissue, is in agreement with our findings droperoxides. Accordingly, it was evident that the drastic fall and supports the results of the present study. It appears likely by over 90% in the ratio of linolenic/linoleic acid in ST pulp, that the manifestation of large air spaces and starch grains strongly signified the breakdown of membrane structure and interspersed among the mass of broken cell walls in the a loss of membrane fluidity (Girotti 1990). The free fatty advanced stage of the spongy tissue disorder could occur, acids liberated during ST development in the pulp also act as possibly, by a reduction in the activity of amylase in pulp substrates for lipid peroxidation and as uncouplers of mito- following the death of cells. chondrial electron transport chain (Skulachev 1998), thus Summing up, the present results have established that a accelerating the pace of cell damage and death. Increased greatly increased competition among fruits for assimilate lipid peroxidation is reported to lead to an increase of elec- supply at 70% fruit maturity significantly decreased the rate trolyte leakage (Kepler and Novacky 1986)dueto of fat synthesis in both pulp and seed of weak sinks.

J. Biosci. 40(2), June 2015 384 S Shivashankar, M Sumathi and TK Roy

Figure 4. Changes in linolenic/linoleic acid ratio (A), MDA content (B), EC (C) and pH (D) of healthy (H), apparently healthy (AH) and ST- affected Alphonso mango pulp (*P≤0.05,**P≤0.005). ST-affected pulp, pulp surrounding the ST-affected portion remaining free of ST symptoms and pulp from healthy fruit were analyzed. Note the drastic fall in the ratio of linolenic acid to linoleic acid, increased accumilation of MDA associated with an increase in the EC. and a fall in the pH of the ST pulp compared to H pulp signifying membrane damage in ST pulp.

Figure 5. Levels of ABA, Z, ZR and iP (A) and GA, DHZR and iPA (B) in seed of healthy and ST-affected Alphonso mango fruit (**P≤0.005). Tr refers to traces. Seed hormones in healthy and ST fruit were analysed by HPLC. Note the rapid increase in the levels of the cytokinins (Z, ZR, iP, DHZR and iPA), gibberellin (GA) and a drop in the levels of ABA in ST seed compared to H seed signifying the initiation of seed germination events in ST seed.

J. Biosci. 40(2), June 2015 Seed VLCFAs and spongy tissue development 385

Figure 6. Proposed biochemical sequence of events leading to spongy tissue development in Alphonso mango fruit. The reduced rate of synthesis of VLCFAs in weak sinks leads to cytokinin production, which triggers seed germination followed by subsequent changes in pulp culminating in the formation of spongy tissue.

Following this, the changes occurring in the profile of fatty acids HL Jayaram, Chief Technical Officer, for help in the analysis and the reduced synthesis of the two VLCFAs, tetracosanoic of seed hormones. acid and hexacosanoic acid, in seed led to a rise in the level of cytokinin, resulting in initiation of seed germination events in the maturing fruit. The sustained flow of pulp moisture to the germinating seed created water-deficit stress in the pulp followed References by hypoxia leading to the excessive production of free radicals (Chirkova et al. 1998). Free radicals then acted upon the fatty acid components of cell membranes, leading to peroxidation of Abou-Aziz AB, Rizk AM, Hammouda FM and El-Tanahy MM 1973 Seasonal changes of lipids and fatty acids in two varieties unsaturated fatty acids. In this process, linolenic acid was more of avocado pear fruits. Plant. Mater. Veg. Qual. XXII 253–259 rapidly degraded compared to linoleic acid, and as a result, the Bach L and Faure JD 2010 Role of very-long-chain fatty acids in ratio of linolenic/linoleic acid was significantly reduced, thus plant development, when chain length does matter. C. R. Biol. decreasing membrane fluidity. This resulted in membrane injury, 333 361–370 tissuedamage(Porteret al. 1995; Gutteridge 1995)andcell Bach L, Michaelson L, Haslam R, Bellec Y, Gissot L, Marion J, Da death, eventually leading to spongy tissue formation (figure 6). Costa M, Boutin J-P, et al. 2008 The plant very long chain Thus, the present study has shown, for the first time, that a hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential reduced synthesis of VLCFAs in the seed during the pre- and limiting for plant development. Proc. Natl. Acad. Sci. USA harvest fruit growth phase may provide the primary trigger for 105 14727–14731 seed germination as demonstrated in Arabidopsis by Nobusawa Bangham AD 1975 Models of cell membranes. In cell membranes: et al. (2013b), thus setting in motion the initiation of spongy Biochemistry, cell biology and pathology, Hospital Practice, New York. Nature 247 438–441 tissue development in the Alphonso mango fruit. Incidentally, Baqui SM, Mattoo AK and Modi VV 1974 Mitochondrial enzymes reduced synthesis of VLCFAs in some seeds may also help in mango fruit during ripening. Phytochem. 13 2049–2055 explain the occurrence of vivipary in Alphonso mango. Bernfeld P 1955 Amylases; in Methods in enzymology, Vol I (eds) NO Kaplan and SP Colowick (NY, USA: Academic Press) pp 149–158 Acknowledgements Bertin N 1995 Competition for assimilates and fruit position affect fruit set in indeterminate greenhouse tomato. Ann. Bot. 75 55–65 We thank the Director, Indian Institute of Horticultural Re- Blokhina O, Violainen E and Fagerstedt KV 2003 Antioxidants, search, Bangalore for providing access to facilities. Grateful oxidative damage and oxygen deprivation stress: a review. Ann. thanks are due to Dr KK Upreti, Principal Scientist, and Mr Bot. 91 179–194

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MS received 12 August 2014; accepted 24 February 2015

Corresponding editor: MAN MOHAN JOHRI

J. Biosci. 40(2), June 2015